Summary of work: Genotoxic/oxidative stresses contribute to the development of degenerative diseases, and may underlie aging itself. Cells respond to such stresses with changes in gene expression, but much remains to be learned about the signaling pathways mediating these effects and their functional significance. This project encompasses studies related to these cellular responses including the following: (1) Activation of Akt by Oxidative Stress. We have demonstrated that hydrogen peroxide treatment results in the activation of a downstream mediator of PI-3 kinase signaling, Akt, and have provided evidence that elevated Akt activity favors survival of hydrogen peroxide-treated cells. (2) Role of Posttranscriptional Events in the Reglation of Cyclin D1 Expression Following Stress. We have demonstrated that cyclin D1 expression is greatly reduced following treatment with many stressful agents including the cyclopentenone prostaglandin PGA2. Exploring the cause for this down-regulation we have found that it occurs largely through posttranscriptional events leading to reduced stability of cyclin D1 mRNA and involves the RNA binding protein AUF1. (3) Role of c-Jun in Influencing Cell Survival Following Stress. We are employing cell lines which either lack c-Jun expression or harbor a dominant negative mutant form of c-Jun (which can not be phosphorylated and activated) to test the role of this transcription factor in influencing cell surival following different stresses. Our results indicate that c-Jun deficiency sensitizes cells to DNA damaging stresses (UVC irradiation, x-irradiation, etoposide) but does not affect sensitivity to other non-DNA damaging inducers of apoptosis (tunicamycin, taxol, thapsigargin). Current studies are exploring the basis for this differential sensitivity. (4) Mechanisms of Singlet Oxygen-Induced Apoptosis. We have recently begun studies to investigate how singlet oxygen leads to death of skin fibroblasts. Preliminary findings suggest the involvment of mechanisms distinct from those mediating death in response to hydrogen peroxide and other models of oxidant injury. (5) Mechanisms Contributing to Age-related Declines in Stress Signaling Pathways. Using primary rat hepatocytes derived from young and aged hosts we have found that the activities of a number of signaling pathways (heat shock response, ERK MAPK activation and p70 S6Kinase) are attenuted in aged cells. This study focuses on the biochemical and molecular bases for this decline. - genotoxic/oxidative stress, PI-3 kinase, ERK, Akt, mRNA stability, cyclin D, c-jun, hepatocytes, singlet oxygen, aging